1. Field of the Invention
This invention relates to a system and method for quantifying by computer image analysis relatively smaller enclosed objects within relatively larger objects. More particularly, this invention relates to a system and method for quantifying macrophage phagocytosis by computer image analysis, and to a method of utilizing the system and method of this invention to evaluate the pathophysiologic, and/or therapeutic effects of any agent by quantifying their endocytic functions and morphological effects.
2. Description of the Prior Art
Generally, phagocytosis is the most important defense mechanism in all phyla of the animal kingdom [van Oss, CJ. Methods of Enzymology,132,3, 1986]. It has an essential role in host defense mechanism against bacterial and/or viral infection(s) [Walters and Papadimitriou, 1978; van Oss, CJ. et al., Phagocytic Engulfment and Cell Adhesiveness, 1975], and the removal of foreign objects, intact red cells and/or red cells that have been fragmented, hemolyzed or are in the process of hemolysis. Increased susceptibility to infection has been reported [Bjorksten, B, and Quie, PG. Abnormalities of Circulating Phagocyte Function, p.181, 1977; Holmes, B. et al., J. Clin. Inv. 46, 1422, 1976] to be associated with defective phagocytic function. Since the phenomenon of phagocytosis was first reported by Haeckel in 1862, several methods for quantitative study of phagocytosis, which can be divided into direct and indirect methods, have been reported, and recently reviewed [van Oss, CJ. Methods of Enzymology, 132,3 1986]. They include microscopic methods, isotopic labeling methods, extraction methods, unphagocytized test particles removal, and measurement of glycolysis, degranulation and respiration activity burst. Current single cell methods for quantifying phagocytosis are either direct microscopic examination [Dunn, PA. et al., J. Immunol. Methods, 64, 71 1983] or by flow cytometric analysis [Absolom, DR, Methods of Enzymology, 132, 95 1986; Bjerknes, B. et al., Rev. Infect. Dis., 11, 16, 1989; Verhoef, J. and Waldvogel, Eur. J. Clin. Microbiol., 4, 379 1985]. Light microscopic examination and manual counting of particles in individual cells is the most common method of quantifying phagocytosis, but only a few cells can be analyzed and no quantitative morphometric data is obtained. Flow cytometry can quantify phagocytosis of many cells in suspension [Dunn, PA and Tyrer, HW, J. Lab. Clin. Med., 98, 374 1981; Steinkamp, JA et al., Science, 215, 64 1982], but cannot provide detailed morphometric data. Besides, the process of most of these methods is time consuming, some are relatively unsafe, and the microscopic methods, in particular, in addition to time consuming and labor intensive, is subjective, only approximate and statistically weak. The inadequacy of these methods has been emphasized in several papers.
Also, computerized systems for quantifying of morphologic and physiologic parameters related to phagocytosis, e.g. intracellular enzymes and ions contents, cell migration, cell surface areas, etc., have recently been reported (Goldstein, E. et al., Reviews Infect. Dis., 10, 92 1988; Askey, DB and Herman, IM, Comput. Biomed. Res., 21, 6, 551 1988). However, intracellular enzymes and ions contents are only indirect methods for the determination of phagocytic function(s). Although cell migration and surface areas may be indicative of the occurrence of infection, they do not provide information needed to evaluate the physiologic, and/or therapeutic effects of agents on phagocytic functions. Furthermore, cytospectrophotometric and image analysis systems which aid non-cumbersome measurement of cellular enzymatic activities and/or functions [Goldstein, E. et al., Reviews Infect. Dis., 10, 92, 1988; Black, CM. et al., Infect. & Immunity, 54, 917, 1986; Donovan, RM and Goldstein, EA, J. Histochem. Cytochem., 33, 551, 1985; Black, CM. et al., J. Infect. Dis., 148, 117, 1983; Goldstein, E. et al., J. Infect Dis., 138, 299, 1978] and quantify morphologic parameters [Hoshino, K, J. Histochem. Cytochem., 31, 1A Suppl, 1983; Erhardt, R. et al., Anal. Quant. Cyto. Histol., 2, 25, 1980] have been produced by the application of computer (first essentially through software engineering, now being improved by both hardware and software developments) and electronics technologies. Although the applications of these systems permit fast and accurate analyses of large population in such a way which would have been impossible using non-computerized microscopic methods, they can not be used for direct quantitative analysis of phagocytic functions.
The lack of computerized quantifying of phagocytic functions has thus far impeded phagocytosis studies involving large number of cells leading to accurate statistical analysis.
The following are the references referred to above by the name of of the author and below by reference number. These references provide further background information to the invention or the description of specific techniques which are incorporated in this application by reference to the extent needed.